1. Field of the Invention
The present invention provides novel compounds comprising phosphoinositides and analogues tagged with stable or radioactive isotopes, novel methods for their preparation by syntheses, and novel key intermediates of synthesis; the novel methods of synthesis are applied also for the preparation of the phosphoinositides in non-labelled form.
2. Related Art
Several phosphoinositides of eukaryotic cells, including 1D-1-(1′-O-fattyacyl′-2′-O-fattyacyl″-sn-glycero-3′-phospho)-myo-inositol (phosphatidylinositol, PtdIns), phosphatidylinositol-4-phosphate (PtdIns-4-P), and phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2) have been well known as metabolically vital lipid precursors of the intracellular second messengers 1D-myo-inositol-1,4,5-trisphosphate and 1′,2′-diacyl-sn-glycerol (see Berridge, M. J. 1987, Annu. Rev. Biochem., 56: 59). Recently, the D-3-phosphorylated phosphoinositides, including the phosphatidylinositol 3-phosphate (PtdIns-3-P), phosphatidylinositol 3,4-bisphosphate (PtdIns-3,4-P2), phosphatidylinositol 3,5-bisphosphate (PtdIns-3,5-P2), phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P3) have been encountered in eukaryotic cells (Whitman, M., et al., 1987, Biochem. J., 247: 165; Whitman, M., et al., 1988, Nature, 332: 644). and, recognized as intracellular messengers (Stephens, L., et al., 1993, J. Biol. Chem., 268: 17162; Duckworth, B. C. and Cantley, L. C. Lipid Second Messengers—Handbook of Lipid Research; Plenum Press: New York, N.Y. 1996, Vol 8, pp 125–175.). Thus the phosphoinositides, and their metabolites, regulate vital biological signaling, and as such, are important materials for research studies, diagnostics reagents, and biotechnology aids. The various enzyme systems involved in the signal transduction via the phosphoinositides, especially the phosphoinositide-specific lipases A, C and D, the phosphoinositide kinases, and phosphoinositide-phosphate phosphatases regulate vital metabolic and physiological processes including cell division, growth and apoptosis. Therefore, phosphoinositides and analogues are being studied for the development of new drug modalities for aberrant signaling including some types of cancer (Kozikowski, A. P. et al., 1993, U.S. Pat. No. 5,227,508).
The phosphoinositides are extremely minor components of plasma and nuclear membranes of cells. Small quantities of PtdIns, PtdIns-4-P, and PtdIns-4,5-P2 can be obtained from natural sources, such as bovine brains, but the D-3-phosphorylated types are not available. The isolated materials are mixtures of molecular species differing in the nature and proportion of the integral fattyacyl ester residues. Individual molecular species with specified fattyacyls or equivalent are required as biochemical research reagents and must be prepared by synthesis. The utility of biochemical research reagent is enhanced by tagging the molecule with isotope labels, including radioactive atom labels, and phosphoinositides with such labels are useful materials.
In the prior art, labelled phosphoinositides have been prepared from tritium labelled myo-inositol using the biochemical machinery of intact cells; alternatively, the biochemical reaction of 32P labelled ATP catalyzed by PtdIns kinase enzymes has been used to introduce an additional phosphate albeit with 32P into bovine brain derived phosphoinositides (see Stephens, L., et al., 1993, J. Biol. Chem., 268: 17162; Duckworth, B. C. and Cantley, L. C. Lipid Second Messengers—Handbook of Lipid Research; Plenum Press: New York, N.Y. 1996, Vol 8, pp 125–175). The routes are inefficient, limited to minute quantities, and in case of kinase enzymes are practical only in the very few laboratories with access to these enzymes. All labelled phosphoinositides so produced are mixtures of molecular species differing in the nature and proportions of the integral fattyacyl ester residues; individual molecular species must be prepared by synthesis, and appropriate synthetic methods are not available.
Only one chemical route has been described whereby a diether analogue of PtdIns-4,5-P2 labelled with tritium was obtained; herein, the tritium label was introduced in the alkyl-ether chain by metal catalyzed reduction of a C—C double bond with tritium gas (Chen, J. and Prestwich, G. D., 1996, J. Labelled Compounds and Radiopharmaceuticals, 39: 251–258). The method is not applicable to phosphoinositides with (poly)unsaturated fattyacyls, and for labelling at other locations, particularly in the inositol and glycerol residues; overall the method is inadequate.
Several chemical syntheses of the phosphoinositides without isotope labels have been described (for example, Aneja, S. G., et al., 1997, Tetrahedron Lett., 38: 803; Bruzik, K. S. and Kubiak, R. J. 1995, Tetrahedron Lett., 36: 2415; Chen, J., et al., 1996, J. Org. Chem., 91: 6305; Desai, T., et al., 1996, Special Publication—Royal Society of Chemistry, 180: 67; Gaffney, P. R. J. and Reese, C. B., 1997, Bioorg. Med. Chem. Lett., 7: 3171; Gou, D.-M. and Chen, C.-S., 1994, J. Chem. Soc., Chem. Commun., 2125; Grove, S. J. A., et al., 1997, J. Chem. Soc., Chem. Commun., 1635; Toker, A., et al., 1994, J. Biol. Chem., 269: 32358; Watanabe, Y., et al., 1994, Tetrahedron Lett., 35: 123; Watanabe, Y., et al., 1995, Tetrahedron, 51: 8969; Watanabe, Y. and Nakatomi, M., 1998, Tetrahedron Lett., 39: 1583). Most of these syntheses are applicable only to analogues with saturated fattyacyls at sn-glycero-1′,2′-O locations. The two most recent (Gaffney and Reese, 1997; Watanabe and Nakatomi, 1998) address the synthesis of phosphoinositides with unsaturated fattyacyls. As mentioned, these methods are not suitable for labelling phosphoinositides, and have not been so applied.